Nitric Oxide Synthase Isoform Protein Research Articles

Ventilatory muscle activation and inflammation: cytokines, reactive oxygen species, and nitric oxide

Strenuous diaphragmatic contractions that are induced by inspiratory resistive breathing initiate an inflammatory response that involves the elevation of pro- and anti-inflammatory cytokines within the diaphragm, which may then spill into the circulation. The production of reactive oxygen species within working respiratory muscles increases in response to these strenuous diaphragmatic contractions. At the same time, diaphragmatic nitric oxide (NO) production declines significantly, despite a time-dependent increase in NO synthase isoform protein expression. The increase in adhesion molecule expression and infiltration of granulocytes and macrophages that follows may contribute to the contraction-induced diaphragm injury. Enhanced generation of reactive oxygen species, oxidative stress augmentation, reduced NO production, and glycogen depletion are potential stimuli for the cytokine induction that is secondary to strenuous diaphragmatic contractions. This production of cytokines within the diaphragm may contribute to the diaphragmatic muscle fiber injury that occurs with strenuous contractions or to the expected repair process. TNF-alpha is a cytokine that compromises diaphragmatic contractility and may contribute to muscle wasting. IL-6 is a cytokine that may have beneficial systemic effects by mobilizing glucose from the liver and free fatty acids from the adipose tissue and providing them to the strenuously working respiratory muscles. Thus cytokine upregulation within the working diaphragm may be adaptive and maladaptive.

Nitric oxide synthesis and oxidative stress in the renal cortex of rats with diabetes mellitus.

Experiments were performed to test the hypothesis that diabetes mellitus disrupts the balance between synthesis and degradation of nitric oxide (NO) in the renal cortex. Diabetes was induced by injection of streptozotocin, and sufficient insulin was provided to maintain moderate hyperglycemia for the ensuing 2 wk. Despite an 80% increase in total NO synthase activity measured by L-citrulline assay, nicotinamide adenine dinucleotide phosphate-diaphorase staining was unaltered, and no changes in NO synthase isoform protein levels or their distribution were evident in renal cortex from diabetic rats. Superoxide anion production was accelerated twofold in renal cortical slices from diabetic rats, with an associated 50% increase in superoxide dismutase activity. Western blots prepared by use of a monoclonal antinitrotyrosine antibody revealed an approximately 70-kD protein in renal cortex from sham rats, the nitrotyrosine content of which was threefold greater in cortical samples from diabetic rats. These observations indicate that the early stage of diabetes mellitus provokes accelerated renal cortical superoxide anion production in a setting of normal or increased NO production. This situation can be expected to promote peroxynitrite formation, resulting in the tyrosine nitration of a single protein of unknown identity, as well as a decline in the bioavailability of NO. These events are consistent with the postulate that oxidative stress promotes NO degradation in the renal cortex during the early stage of diabetes mellitus.

Differential expression of nitric oxide synthase isoforms in form-deprived chick eyes

Purpose. To clarify whether nitric oxide synthase (NOS) is involved in development of myopia, we examined the influence of form deprivation on the expressions of NOS isoform mRNA.Methods. NOS isoform cDNAs were amplified from total RNA extracted from control and 7-day-form-deprived chick retina-RPE (retinal pigment epithelium)-choroid, using competitive RT-PCR (reverse-transcription polymerase chain reaction). Each NOS isoform protein was also analyzed by Western blotting and immunohistochemistry.Result. Expression of inducible NOS (iNOS) mRNA was highest in the control chick retina-RPE-choroid, followed by the expression of brain NOS (bNOS) mRNA. Expression of endothelial NOS (eNOS) mRNA was faint. The iNOS protein level, however, was only slightly higher than the levels of the bNOS and eNOS proteins and was found mainly in the outer part of the photoreceptor layer and inner and outer parts of RPE and choroid. bNOS alone was found in the outer nuclear layer. Although form deprivation reduced the iNOS and bNOS mRNA expressions, only the iNOS protein showed significant reduction.Conclusion. All three NOS isoforms were expressed in chick retina-RPE-choroid. Predominant expression of iNOS, instead of bNOS and eNOS, suggested the existence of ocular tissue-specific regulation of the iNOS gene. In addition to differences in expression level, bNOS displayed regional differential expression. Moreover, only iNOS was reduced in response to form deprivation. It is suggested that NOS isoforms may be differentially involved in the mechanisms regulating the posterior eye tissues, including myopic eye growth. Curr. Eye Res. 17: 586–593, 1998.